This invention relates to a device for performing immunoassays to detect a variety of antigens in a fluid sample. More particularly, this invention relates to a novel means for producing a defined reactive zone within a device and controlling reagent flow therein. It is believed that this is the first immunoassay device that employs a single porous member as both an antibody/reagent support and as a spent reagent reservoir and does not require external means to direct the flow of sample and reagents within the device.
Antibody-mediated analyses are widely utilized for the estimation of antigens in a liquid sample. Once an antibody has been developed against an antigen, the antibody can be employed as a reagent for detection of the antigen. Increasingly, samples such as serum, urine, other biological fluids and industrial fluids are analyzed using several different antibodies raised against various antigens to fully characterize the antigen make-up of the sample.
The practice of using antibodies to detect soluble antigens is well known in the art. In general, an antibody directed against an antigen of interest is immobilized on an insoluble substrate and exposed to a sample which may contain the antigen. If the sample contains the antigen, an antibody/antigen complex will form which can be detected by various means including but not limited to colorimetric, radiometric, turbidimetric and fluorometric immunoassay procedures.
The variety of apparatus and methods for controlling antibody-mediated reactions is the subject of a diverse technology. See, P. Tijssen, PRACTICE AND THEORY OF ENZYME IMMUNOASSAYS, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY (1985). Conventional immunoassay procedures employ antibody immobilized as a coating on plastic or glass tubes. Formation of an antibody/antigen complex and the detection thereof typically require a series of incubation and rinsing steps. While effective, the method described above is substantially time consuming.
More recently a variety of strategies have been developed to form and detect antibody/antigen complexes within a single-use, disposable assay device utilizing porous antibody/reagent supports. Typical of this technology is U.S. Pat. No. 4,399,229 (Kelton et al.) in which the antibody/reagent support consists of a bacterium to which an antibody is bound, the bacterium being entrapped in a glass fiber filter. In U.S. Pat. No. 4,632,901 (Valkirs et al.), an antibody is bound to a permeable membrane which is contacted by a porous capillary member which directs the flow of sample and reagents within the device. U.S. Pat. No. 4,655,034 (Chandler) discloses a reaction chamber which employs a plurality of syringes to direct the flow of fluids within the chamber.
The foregoing devices generally require more than one member: a first member to which antibody is bound and which serves to immobilize the antibody; and at least a second member which serves as (i) a means for directing the flow of fluid within the device by capillary action or channeling and/or, (ii) as a spent fluid reservoir. In addition, these devices require a variety of means to direct sample and reagents therein such as positive pressure, vacuum and centrifugation.
Moreover, the foregoing devices allow sample and reagents to flow in both a downward and a lateral direction. Lateral reagent flow contributes to a higher incidence of inaccurate results due to the tendency of spent reagents to accumulate at the periphery of the antibody/antigen reaction zone. These reagents tend to interact and produce color reactions that are easily mistaken for a true positive or negative result.
A need therefore continues to exist for a simple immunoassay device for use in procedures to detect one or more analytes comprising a single porous material that can serve as both an antibody/reagent support and as a spent reagent reservoir. In addition, a need continues to exist for a device comprising a means for directing the flow of sample and reagents within the device in a manner that will eliminate both lateral diffusion and backflow of reagents without the necessity of additional external means.